Genetic characteristics influence the phenotype of marine macroalga Fucus vesiculosus (Phaeophyceae)

Abstract Intraspecific variation is an important component of heterogeneity in biological systems that can manifest at the genotypic and phenotypic level. This study investigates the influence of genetic characteristics on the phenotype of free‐living Fucus vesiculosus using traditional morphological measures and microsatellite genotyping. Two sympatric morphotypes were observed to be significantly genetically and morphologically differentiated despite experiencing analogous local environmental conditions; indicating a genetic element to F. vesiculosus morphology. Additionally, the observed intraclonal variation established divergent morphology within some genets. This demonstrated that clonal lineages have the ability to alter morphological traits by either a plastic response or somatic mutations. We provide support for the potential occurrence of the Gigas effect (cellular/organ enlargement through genome duplication) in the Fucus genus, with polyploidization appearing to correlate with a general increase in the size of morphological features. Phenotypic traits, as designated by morphology within the study, of F. vesiculosus are partially controlled by the genetic characteristics of the thalli. This study suggests that largely asexually reproducing algal populations may have the potential to adapt to changing environmental conditions through genome changes or phenotypic plasticity.

Differences in genotype and phenotype characterize the intraspecific variation of a species. There is growing recognition that intraspecific variation plays an important role in biological systems (Bolnick et al., 2011;Violle et al., 2012). For example, intraspecific phenotypic variation can influence plant-herbivore interactions (Cruz-Rivera & Friedlander, 2013), while greater intraspecific genetic variation in key species has been shown to increase the complexity of the associated food web (Barbour et al., 2016). Consequently, the genotypic and phenotypic complexity of a species can have ecosystem-level effects. Within a species, genotypes and phenotypes are disparate. Many genotypes may encode the same phenotype, while the number of genotypes encoding each phenotype is also unevenly distributed (Catalán et al., 2018). Consequently, potential complexity is controlled by the specific biological properties of a species.
Although typically associated with hard substratum as an attached form, F. vesiculosus can also be frequently found in a free-living form lying unattached on any substratum. This form is particularly common throughout the Baltic Sea (Bauch, 1954;Luther, 1981;Luther et al., 1975;Meyer et al., 2019;. Free-living populations are known to display vast morphological variation, as determined by several form series (Kjellman, 1890;Svedelius, 1901). Here, we investigate the role of genetic factors on phenotypic variation in several free-living F. vesiculosus populations within the Baltic Sea. We focus on morphological divergence and how genotypic factors influence this. Atypically for F. vesiculosus, the Baltic Sea distribution demonstrates facultative asexuality (Ardehed et al., 2016;Johannesson et al., 2011;Pereyra et al., 2013;Tatarenkov et al., 2005). Asexual reproduction, presumably by means of fragmentation and/or adventitious branches, is particularly pervasive within the free-living form, although the prevalence of clonality is highly variable among populations .
Accordingly, free-living populations provide an ideal study system as they can consist of varying proportions of clones, either from single or multiple lineages, and unique multilocus genotypes (MLGs) . Each clone represents a physiological individual (ramet), collectively all ramets originate from a single zygote (genet) and are genetically identical barring somatic mutations (Harper, 1977;Jackson & Coates, 1986;Pan & Price, 2002).
Consequently, the occurrence of multiple ramets can provide a novel opportunity to explore aspects of morphological traits.
Polyploidy (whole-genome replication) can affect phenotype, as has been frequently observed in plants (Levin, 1983;Soltis et al., 2014). In plants, polyploidy is typically associated with larger cell sizes compared to diploidy (Müntzing, 1936;Ramsey & Ramsey, 2014;Stebbins, 1971), which in turn correlates with larger adult sizes (Čertner et al., 2019;Te Beest et al., 2012). Unattached Fucus spp. are known to represent varying ploidy levels, with both allopolyploidy (hybridization between two or more related species) and autopolyploidy (multiplication of the whole genome of a single parent species) having been observed in natural populations (Coyer et al., 2006;Sjøtun et al., 2017). Recent investigations indicate that polyploidization may also occur within free-living F. vesiculosus within the Baltic Sea . However, polyploidy in Fucus spp. has typically been associated with miniaturization of the thalli (e.g., F. cottonii (Sjøtun et al., 2017) and "muscoides-like" Fucus (Coyer et al., 2006)). Thus, the current anecdotal observations of the correlation between ploidy and morphology in Fucus spp. are in opposition to the more widely accepted understanding within plants.
By definition, phenotypic plasticity construes that one genotype has the ability to express a multitude of phenotypes in response to biotic and abiotic conditions (Bradshaw, 1965). Accordingly, species characterized by a high degree of phenotypic plasticity have the ability to express a large range of phenotypes from single genotypes. The effects of phenotypic plasticity are largely underappreciated when it comes to diversification, yet they can greatly impact intraspecific variation (Pfennig et al., 2010) including morphological aspects of an organism's phenotype (Sommer, 2020). Phenotypic plasticity can also effect ecosystems at varying levels, having a variety of direct and indirect interactions at the individual, population, and community level (Miner et al., 2005). In clonal populations, phenotypic plasticity represents an ability for a genet to adapt to diverse environmental conditions (Bruno & Edmunds, 1997;Geng et al., 2016).
Whether dispersed or in close proximity, ramets experience different selective pressures as a result of varying biotic and abiotic conditions. Phenotypic plasticity can allow an adaptive response to these variable conditions in the absence of recombination, allowing the persistence of the genet. The resulting plastic responses produce intraclonal variability, creating an additional level of complexity within clonal lineages. Intraclonal variation can also be the result of genome changes during somatic growth (Santelices, 2004;Santelices & Varela, 1993). Somatic mutations, genetic changes that occur during mitosis, can be a result of internal or external environmental factors such as temperature stress, cell age, and oxidative stress (Schoen & Schultz, 2019). Often neutral or beneficial, somatic mutations may result in phenotypic changes, as seen by their frequent occurrence in commercially important plant varieties (Tilney-Bassett, 1986).
These mutations can accumulate, with the potential to be inherited by progeny or propagated in ramets. The accumulation of somatic mutations can result in genetic mosaicism, which in clonal lineages can lead to the formation of genotypically diverse, independent ramets (Gill et al., 1995). As somatic mutations are potentially heritable, there are consequences for evolutionary processes and diversification in clonal lineages that otherwise have limited recombination (Gross et al., 2012;Klekowski, 1988). Clonal lineages can therefore represent a mosaic of phenotypes due to either potentially reversible plastic responses or genetically fixed genome changes. Overall intraclonal variation can promote adaptive responses and potential genet persistence in changing environments for facultative asexual species, such as F. vesiculosus, as well as acting as a potential source of intraspecific complexity. As a highly plastic species with persistent clonality, we expect to observe intraclonal morphological variation within free-living F. vesiculosus clonal lineages.
Overall, this study aims to examine the influence of genotype and polyploidy on morphological traits of free-living F. vesiculosus at the intraspecific and intraclonal level.

| Sampling
Sampling was performed by SCUBA diving in June 2019 at Askö  Figure 1). All sites were within close proximity of the shore, in shallow, sheltered embayments associated with Phragmites australis reed beds (Data S1).
The bottoms at all sites were soft, being either muddy or sandy substrata. Sampling depth ranged between 1.5 and 3.4 m, and the salinity ranged from c.5.9 to 6.1. Free-living F. vesiculosus was the dominant macroalga within these locations. At sites AS1 and TZ1, the thalli were entangled within P. australis. At each site, four 20 × 20 cm frames were randomly placed on the seafloor with all vegetation within the frame being collected into net bags. If available, up to five F. vesiculosus thalli per frame (n = 115) were selected for further measurements. In frames with more than five thalli, all other collected thalli (excluding the selected five per frame) were discarded from further analysis. All detached thalli were treated as separate physiological individual thalli for means of morphological analysis and genotyping. If fewer than five separate thalli were found within the plot, then the maximum number of separate thalli available was chosen.

| Morphological measurements
The 115 collected thalli (max 5 per frame) underwent morphological analysis. Due to the free-living nature of the F. vesiculosus thalli (i.e., lack of holdfast), many of the standard morphological measures normally used for Fucus spp. are inappropriate (e.g., stipe length, stipe width, and distance of dichotomies). Consequently, three standard measures not reliant on the presence of a holdfast were recorded.
These measures were thallus height (cm), wet weight (g), and five repeats of thallus width (cm) per individual sample (Ruuskanen & Bäck, 1999. Thallus height was determined as the distance from oldest growth to the longest apical tip, while thallus width was the width across the thallus 5 cm from the apical tip. Site AS2 displayed two distinct morphotypes coexisting with no perceivable abiotic barriers (Data S2). Within this study, morphotype was defined as a group of thalli with similar morphology using our defined morphological measures. Samples were taken from both morphotypes and defined as AS2_N (narrow morphotype) and AS2_T (typical morphotype). The distinction between the morphotypes was evident through visual inspection, but to ensure an accurate definition, the narrow morphotype was defined as an average thallus width < 1 cm, whereas the typical morphotype was an average thallus width > 1 cm. Measurements for the two morphotypes fell within either category with no intermediates.

| DNA extraction and microsatellite genotyping
All collected thalli were also genotyped (n = 115). For a detailed description of the microsatellite genotyping protocol, see  (including Supplementary Materials).
Microsatellite genotyping followed the aforementioned protocol exactly, although a brief summary is provided herein and in Table S1.

| Data analysis
Alleles were scored using GeneMapper 5 (Applied Biosystems™) and peaks within polyploid samples were determined to be accurate when they could be observed consistently in PCR reactions and also represented similar peak signatures to those observed in diploid samples. The majority of alleles determined in polyploid samples were also common throughout the diploid samples suggesting that spurious amplifications are unlikely. The assumption that the observation of ≥3 alleles is a result of polyploidy is supported within Fucus by Coyer et al. (2006). Of 115 samples, 50 were determined to have ≥3 alleles in at least one locus, inferring a potentially polyploid sample. Assigning the allele dosage of an apparent polyploid sample is problematic using microsatellite genotyping as the marker phenotype can represent multiple genotypes. For example, a triploid sample with a marker phenotype of AB may have a genotype of AAB or ABB. Consequently, we used the software Genodive version 3.05 (Meirmans, 2020) to perform data correction as in . All genotyping data analyses referenced herein were performed on Genodive version 3.05 (Meirmans, 2020) unless expressly mentioned. Clonal assignment was performed using a Stepwise Mutation Model with a threshold of 0 and allocating clones specific to population. A total of 68 genotypes were assigned from 115 samples. The proportion of clones and ploidy levels were calculated manually. Pairwise population differentiation was tested using the test statistic Rho (Ronfort et al., 1998)

| Genetic structure of populations
The total number of genotypes at each site ranged from 4 to 19 (Table 1) (Table 2).  (Table S3) Table 2). As previously mentioned, these morphotypes were easily identifiable through visual inspection, yet we also confirm that the morphological divergence was statistically significant (Table S4a) Table S4b). Therefore, the genetic similarity between the sites is mirrored in the morphological similarity.

| Genetic influences on morphology
The data consisted of 65 diploid and 50 polyploid samples.
Correlations were identified between ploidy and morphology with apparent polyploids being significantly larger than their diploid counterparts (Table 3; Figure 3). This is shown by the Mann-Whitney mean ranks, which for all morphological variables were consistently higher in the polyploid grouping, ranging between 40 and 50 for diploids and 68 and 81 for polyploids (Table 3). The enlargement of morphology due to polyploidy is more supported in thallus height (U = 487, p = .000) and thallus width (U = 466, p = .000), while less but still significantly within wet weight (U = 1116, p = .004).
The median thallus heights and widths were significantly dissimilar (p < .001) between ploidies (Figure 3a,c), while the medians for wet weight were more similar (Figure 3b). Diploid thallus height was relativity tightly grouped with several outliers. Polyploids were more loosely grouped with no outliers. The largest thallus height was recorded in a diploid sample; however, this sample was over 20 cm larger than the next largest diploid sample. This outlier does not represent the general trend in diploid thallus height. The maximum thallus height was greater for polyploids, while the minimum for each ploidy was more similar (Figure 3a). For wet weight, both ploidies were relatively tightly grouped, although both have several outliers ( Figure 3b). Again, the largest recorded measure was from a diploid sample outlier. The distribution of diploid samples was skewed toward the lower scale, while polyploids are more equally distributed.
Both ploidy levels displayed equal spread among thallus width, with a large range of widths being observed (Figure 3c). Maximum ranges between ploidies were similar, while minimum were more dissimilar.

| Genotype determines morphology
In F. vesiculosus, genotype has been observed to influence several phenotypic features, including tolerance to warming and acidification (Al-Janabi et al., 2016), resistance and tolerance to fouling (Honkanen & Jormalainen, 2005), and production of antiherbivory compounds (Jormalainen & Ramsay, 2009). Consequently, it is unsurprising that we determine that genotype appears to express control over morphological traits. The corresponding morphological and genetic differentiation at AS2 (i.e., AS2_N and AS2_T) determines that the morphotypes are both morphologically and Ploidy n  (Bauch, 1954;Cotton, 1912;Häyrén, 1949;Luther, 1981;Svedelius, 1901). Thus, these two ecotypes likely originate from two separate populations with distinct environmental conditions and limited connectivity, which force the development of divergent phenotypes and genotypes. The rafting pieces of thalli from each separate population may then migrate to the free-living site, whereby they increase their representation within the site by clonal growth. As free-living forms are surmised to rarely reproduce sexually (Bauch, 1954;Häyrén, 1949;Svedelius, 1901), barriers to gene flow are maintained through strongly restricted recombination induced by high levels of asexual reproduction. Consequently, the two ecotypes coexist within new, neutral environmental conditions, yet morphology is genetically fixed from their population of origin. This means that the two morphotypes are a result of genetic responses to two different habitats, likely geographically distant from AS2, which retain their morphological traits when supplied into the neutral free-living habitat. Overall, the ecotypes are discrete entities with clear differences in morphology and genetics.

Sum of ranks
Further supporting that there may be an underlying genetic basis to expressed morphological traits; thalli at AS2_N and AS3 were genetically and morphologically similar. These sites were composed of varying proportions of clonal and unique MLGs with allele frequencies being remarkable similar, yet no clonal lineages were shared. Despite the geographic distance (4.3 km), these closely related genotypes formed analogous morphotypes, consequently inferring that these genotypes encode the same phenotype. Thus, it appears that genotype exerts some level of control on the morphology of F. vesiculosus.
It is important to note that although genotypes are associated with different phenotypes, the microsatellite markers used within this study are putatively neutral and therefore would be assumed not to be under selection. Consequently, the genotype variants should confer no fitness advantage (Stouthamer & Nunney, 2014).
Thus, microsatellite genotyping can only provide partial insight into the genotype-phenotype relationship. The use of adaptive markers (i.e., genes that directly influence fitness) would be required to determine a direct link between phenotypic features and genotype (Kirk & Freeland, 2011). However, as a nonmodel organism, candidate genes and the phenotypic traits that they influence in natural populations are poorly understood in Fucus species.

| Divergent morphology in genetically identical thalli
The high occurrence of clonal MLGs provides a novel situation, of algae were determined to show plasticity, with inducible defenses from herbivory being the primary cue for triggering plasticity, although observed plasticity in response to the environment is also common (Padilla & Savedo, 2013). Although all F. vesiculosus ramets within each genet were only present in single sites, environmental factors and herbivory are likely to differ across the site as well as each ramet being subjected to differing microenvironments. For example, within the Baltic Sea it is known that the rate of herbivory on F. vesiculosus varies with depth (Jormalainen & Ramsay, 2009).
Consequently, the observed intraclonal variation may be a result of phenotypic plasticity of genetically identical ramets.
The observed intraclonal variation may also be a consequence of genome changes during somatic growth. Intraclonal variation in algae may result from several mitotic processes, including somatic mutations, intragenomic recombination, mobile genetic elements, gene duplication, and ploidy changes (Buss, 1985). As microsatellites do not represent the whole genome, some level of genomic variation within clonal lineages may be masked. Additionally, within algae mutations appear fairly common (Russell, 1986), with some resulting in alterations to the phenotype (Poore & Fagerström, 2000;van der Meer, 1981van der Meer, , 1990van der Meer et al., 1984). Unlike many algae,  (Klekowski, 1988).
However, reproduction through clonal growth poses a far greater chance of preserving somatic mutations. In F. vesiculosus growth occurs through apical cells, whereby if the somatic mutation occurs in an apical cell, the mutant cell genotype will be passed on to all subsequent tissue derived from that cell resulting in genetic mosaicism (Poore & Fagerström, 2000). The resulting changes in morphology will then appear first at the tips of the thallus and as growth progresses a part of the clone develops a different phenotype (Santelices, 2001). After divergent morphological development occurs, the phenotypically varied piece may become detached from the larger thallus and form a new physiologically independent morphological variant (Santelices & Varela, 1993 Villemereuil et al., 2016;Linhart & Grant, 1996).
Classically, clonal organisms have been viewed as more susceptible to detrimental effects caused by changing environmental conditions. However, intraclonal variation among ramets suggests a level of adaptive potential within clonal organisms. Intraclonal variation can increase the possibility of genet survival (Santelices et al., 1995). If morphological variants correspond to improved fitness traits, this allows the genet to be able to adjust to environmental changes improving the persistence of the genet. This would be true of both phenotypic plasticity and somatic mutation. Although this concept may be particularly relevant when considering somatic mutations, as these may be inherited by subsequent generations which then offer potential for adaptive evolution in the absence of recombination (Poore & Fagerström, 2000). However, irrelevant of the true underlying mechanism causing intraclonal variation, the observation indicates that clonal free-living F. vesiculosus of the Baltic Sea has the potential to adapt to changing environmental conditions. Importantly, intraclonal variation poses an ability to increase the complexity in free-living F. vesiculosus despite persistent clonality.
The current understanding of Fucus polyploids, potentially deriving from either allo-or autopolyploidization (Coyer et al., 2006;Sjøtun et al., 2017), confounds our ability to determine the exact processes deriving polyploidy. However, stressful habitats with regard to salinity and temperature, as the Baltic Sea could be considered, have been suggested to facilitate polyploidization in Fucus (Sjøtun et al., 2017).
Further research using appropriate techniques (e.g., microspectrofluorometry or flow cytometry) is needed to assess the accuracy of polyploidy within the Baltic Sea population; however, the genotyping data presented here strongly suggests polyploidization. The paucity of data relating to polyploidization in Fucus spp. limits the ability to assess how polyploidy changes ecological interactions (Segraves, 2017).
However, our observed correlation between morphological traits and ploidy suggests potential consequences at a community level.
Polyploidy results in an increase in cell DNA content which has direct consequences for cell size (Müntzing, 1936;Stebbins, 1971). This occurrence, termed the Gigas effect, results in an increased cell size and consequent increase in organ and plant size (Sattler et al., 2016).
The impacts of the Gigas effect on morphology are well documented in plants (Doyle & Coate, 2019;Knight et al., 2005) with the general trend resulting in polyploids having larger features than their diploid conspecifics (Porturas et al., 2019;Stebbins, 1971). However, a few exceptions in plants have also been documented (Ning et al., 2009;, and the effect of genome size appears weaker at higher levels (e.g., tissue or organs (Knight & Beaulieu, 2008)). These exceptions twinned with the previous anecdotal observation of miniaturization in Fucus polyploids (Coyer et al., 2006;Sjøtun et al., 2017) indicated that Fucus spp. may not follow the Gigas effect. However, our observations invalidate this assumption. As free-living F. vesiculosus frequently reproduces through clonal growth, the Gigas effect may have significant influences in population dynamics by enhancing the establishment success of polyploid genets through facilitating the production of more ramets and increasing ramet establishment and survival rates. Likewise, the Gigas effect can affect interactions with other organisms potentially altering the ecology of these species (Segraves & Anneberg, 2016). The interactions between the Gigas effect and community composition and functioning are likely to be complex, as although the thallus biomass is larger, the growth rate and tissue composition of polyploids will likely be altered as well (Segraves, 2017 We emphasize that rates of polyploidy in our study are likely underestimated. Through using microsatellite genotyping, polyploids may be masked as diploids and similarly tetraploids may be masked as diploids or triploids. In our system, we assumed a tetraploid dominance of polyploids, as is the general consensus in many natural polyploid populations (Comai, 2005). Yet nearly, all polyploid samples appeared triploid. Thus, we suggest that the use of microsatellite markers has hindered the ability to assess levels of polyploidy.
Although this underrepresentation of tetraploids may also translate into an overrepresentation of diploids, we suggest that diploidy is fairly common in the system due to the levels of previous research solely identifying diploidy. Thus, we feel it is appropriate to determine differences between diploids and polyploids, but any comparison between polyploid levels within our study would be erroneous.

ACK N OWLED G M ENTS
We are grateful to Viivi Halonen for her contributions to the fieldwork/sample processing and Susanne Qvarfordt, Oskar Nyberg, and Chiara D'Agata for their assistance with fieldwork at Askö Laboratory. We are also grateful to the staff at the Molecular Ecology and Systematics (MES) laboratory, University of Helsinki, for their assistance with genotyping. We thank Jaanika Blomster for her help in reviewing the manuscript. Funding for this project was provided through grants from the Walter and Andrée de Nottbeck Foundation, Onni Talas Foundation, the Baltic Sea Centre (Askö grants), and the Academician Ilkka Hanski Fund.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.6084/ M9.figshare.19690930.

DATA AVA I L A B I L I T Y S TAT E M E N T
Microsatellite genotyping and morphology data are openly available